ABSTRACT Antisense oligonucleotides (ASOs), siRNA and splice switching oligonucleotides (SSOs) all have immense potential as therapeutic agents, potential that is now being validated as oligonucleotides enter the clinic. SSOs in particular possess great therapeutic flexibility since they can be used to increase, decrease or alter the pattern of mRNA expression. However, progress in oligonucleotide-based therapeutics has been limited by the difficulty in delivering these complex molecules to their sites of action in the cytosol or nucleus of cells within specific tissues. There are two aspects to the delivery problem. The first is that most types of oligonucleotides have poor uptake into non-hepatic tissues such as the lung. The second is that much of the oligonucleotide that is taken up by cells is entrapped in endosomes where it is pharmacologically inert. Initos Pharmaceuticals has developed a dual approach that overcomes both aspects of the oligonucleotide delivery problem, focusing particularly on SSOs. First, we make use of SSOs comprised of peptide-morpholino oligonucleotide conjugates (P-PMOs) that have greater cell uptake and a broader tissue distribution than conventional oligonucleotides. Second, Initos has addressed the intracellular delivery issue by creating novel proprietary oligonucleotide enhancing compounds (OECs) that mobilize all types of oligonucleotides from endosomes thus significantly increasing their therapeutic effects. While the P-PMO/SSO+OEC combination is a platform technology applicable in many therapeutic contexts, this proposal focuses on two challenging problems in pulmonary disease. First, we will address the correction of splicing mutations in cystic fibrosis patients who are refractory to current therapies including modulator drugs. Second, we will use the P-PMO/SSO+OEC combination to reduce the hypersecretion of mucins MUC5AC or MUC5B that is characteristic of several major airway diseases including asthma, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). During our current Phase I STTR we established proof of concept for the P-PMO/SSO+OEC technology by showing: (a) correction of splicing in lungs in a murine reporter model; (b) correction of splicing and restoration of function in CF patient-derived differentiated airway cells; (c) dramatic reduction of MUC5AC expression in human cells. In our Phase II SBIR application we propose IND-facilitating development of our unique P- PMO/SSO+OEC technology. The goal is to identify P-PMO/SSO+OEC entities that are highly efficacious and minimally toxic by optimization of composition, dose, timing and route of administration. The deliverables for the proposed project are the development for pre-IND evaluation of P-PMO/SSO+OEC entities for therapy of a CF splicing mutation and for reduction of MUC5AC or MUC5B hypersecretion.